Peracid-generating compositions

ABSTRACT

Described herein are tooth whitening strips comprising an hydratable adhesive film having a granular bleaching ingredient attached thereto, wherein upon hydration, the granular bleaching ingredient releases hydrogen peroxide which is used by an enzyme catalyst having perhydrolytic activity to enzymatically produce an effective amount of peracid bleaching agent from an acyl donor substrate. Methods of making and use the tooth whitening strips are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 61/577,499,filed on 19 Dec. 2011, which is hereby incorporated by reference in itsentirety.

BACKGROUND

There exists a need for whitening strips suitable for home use, havingreduced total levels of peroxide, yet providing enhanced whiteningactivity.

SUMMARY

The invention provides whitening strips comprising a granular bleachingingredient in combination with an enzyme having perhydrolytic activity(“perhydrolase”) which comprises the conserved structural motif of thecarbohydrate esterase family 7 and an acyl donor, such that upon use,the peroxide released by the granular bleaching ingredient reacts withthe acyl donor in the presence of the perhydrolase to form a peracid, indirect proximity to the teeth, without substantial dilution fromformulation excipients, thereby permitting enhanced bleaching of theteeth with much lower total amounts of peroxide.

The strips comprise an adhesive film, either single layer or multiplelayers (e.g., two layers), which when hydrated with water or salivabecomes sufficiently adhesive to stick to teeth. Granular bleachingingredient is attached to the side of the film to be placed in contactwith the teeth. Upon application, the bleaching ingredient is placeddirectly on the teeth (that is, between the teeth and the adhesivelayer). The granules then release peroxide by rapidly dissolving inwater. The bleaching ingredient can be optionally coated by a quicklydissolving material, such as sodium sulfate, cornstarch or gum Arabic.Optionally, the strips provide a second layer in the film is present toprolong the exposure time. This second layer can be insoluble in water,which would require the user to remove the strip after treatment, orerodible in water, which would cause the strip to dissolve aftersufficient treatment. The strip further comprises a perhydrolase (anenzyme capable of catalyzing the reaction of carboxylic acid ester andhydrogen peroxide to form a peracid), which may also be provided ingranular form on the surface of the film, and an acyl donor, e.g.,selected from carboxylic acids and acyl compounds, for example,triacetin or sorbitol hexaacetate, wherein the acyl donor reacts withthe peroxide source in the strip in the presence of the perhydrolase toform a peracid, which enhances the bleaching action of the strip.

Some embodiments of the present invention provide a tooth whiteningstrip comprising a hydratable adhesive film with a first side and asecond side, the first side having a granular bleaching ingredientattached thereto, wherein the tooth whitening strip further comprises,in or on the film or in the form of granules attached to the first sideof the film;

a) an enzyme having perhydrolytic activity, said enzyme having acarbohydrate esterase family 7 (CE-7) signature motif that aligns with areference sequence SEQ ID NO: 1, said signature motif comprising:

-   -   i) an RGQ motif at positions corresponding to positions 118-120        of SEQ ID NO: 1;    -   ii) a GXSQG motif at positions corresponding to positions        186-190 of SEQ ID NO:1; and    -   iii) an HE motif at positions corresponding to positions 303-304        of SEQ ID NO:1; and

(b) at least one acyl donor substrate, said substrate selected from thegroup consisting of:

-   -   i) esters having the structure

[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O    -   R₆=C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with hydroxyl groups or C1 to C4 alkoxy        groups, wherein R₆ optionally comprises one or more ether        linkages for R₆=C2 to C7;    -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or        a five-membered cyclic heteroaromatic moiety or six-membered        cyclic aromatic or heteroaromatic moiety optionally substituted        with hydroxyl groups; wherein each carbon atom in R₅        individually comprises no more than one hydroxyl group or no        more than one ester group or carboxylic acid group; wherein R₅        optionally comprises one or more ether linkages;    -   M is an integer ranging from 1 to the number of carbon atoms in        R₅; and wherein said esters have solubility in water of at least        5 ppm at 25° C.;    -   ii) glycerides having the structure

-   -   wherein R₁=C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O);    -   iii) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; and    -   iv) acetylated saccharides selected from the group consisting of        acetylated monosaccharides, acetylated disaccharides, and        acetylated polysaccharide; wherein upon hydration of the        hydratable adhesive film hydrogen peroxide is released from the        granular bleaching ingredient and said enzyme catalyzes the        formation of an effective amount of a peracid.

Other embodiments provide of the present invention provide a method ofwhitening teeth comprising providing a packaging system comprising thetooth whitening strip according to any foregoing claim; removing thetooth whitening strip form the packaging system; and contacting thetooth whitening strip directly to the teeth for a period of timesufficient time whiten the teeth; wherein the tooth whitening strip ishydrated by moisture present in the oral cavity or on the tooth surfaceor is hydrated after step (b) but prior to step (c).

Further areas of applicability of the present invention will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating the preferred embodiment of the invention, are intended forpurposes of illustration only and are not intended to limit the scope ofthe invention.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences comply with 37 C.F.R. §§1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EuropeanPatent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules5.2 and 49.5(a-bis), and Section 208 and Annex C of the AdministrativeInstructions. The symbols and format used for nucleotide and amino acidsequence data comply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO: 1 is the amino acid sequence of Thermotoga maritima C277Svariant perhydrolase.

SEQ ID NO: 2 is the amino acid sequence of fusion protein comprising theThermotoga maritima C277S variant perhydrolase coupled to a toothbinding domain (also known as “EZ-7” in International Patent ApplicationPublication No. WO2012/087970A2 to Butterick et al.).

SEQ ID NO: 3 is the nucleic acid sequence encoding a cephalosporin Cdeacetylase from Bacillus subtilis ATCC® 31954™.

SEQ ID NO: 4 is the amino acid sequence of a cephalosporin C deacetylasefrom Bacillus subtilis ATCC® 31954™.

SEQ ID NO: 5 is the amino acid sequence of a cephalosporin C deacetylasefrom Bacillus subtilis subsp. subtilis strain 168.

SEQ ID NO: 6 is the amino acid sequence of a cephalosporin C deacetylasefrom B. subtilis ATCC® 6633™.

SEQ ID NO: 7 is the amino acid sequence of a cephalosporin C deacetylasefrom B. licheniformis ATCC® 14580™.

SEQ ID NO: 8 is the amino acid sequence of an acetyl xylan esterase fromB. pumilus PS213.

SEQ ID NO: 9 is the amino acid sequence of an acetyl xylan esterase fromClostridium thermocellum ATCC® 27405™.

SEQ ID NO: 10 is the amino acid sequence of an acetyl xylan esterasefrom Thermotoga neapolitana.

SEQ ID NO: 11 is the amino acid sequence of an acetyl xylan esterasefrom Thermotoga maritima MSB8.

SEQ ID NO: 12 is the amino acid sequence of an acetyl xylan esterasefrom Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 13 is the amino acid sequence of a cephalosporin Cdeacetylase from Bacillus halodurans C-125.

SEQ ID NO: 14 is the amino acid sequence of a cephalosporin Cdeacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 15 is the amino acid sequence of a Thermotoga neapolitanaacetyl xylan esterase variant from U.S. Patent Application PublicationNo. 2010-0087529 (incorporated herein by reference in its entirety),where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 16 is the amino acid sequence of a Thermotoga maritima MSB8acetyl xylan esterase variant from U.S. Patent Application PublicationNo. 2010-0087529, where the Xaa residue at position 277 is Ala, Val,Ser, or Thr.

SEQ ID NO: 17 is the deduced amino acid sequence of a Thermotogalettingae acetyl xylan esterase variant from U.S. Patent ApplicationPublication No. 2010-0087529, where the Xaa residue at position 277 isAla, Val, Ser, or Thr.

SEQ ID NO: 18 is the amino acid sequence of a Thermotoga petrophilaacetyl xylan esterase variant from U.S. Patent Application PublicationNo. 2010-0087529, where the Xaa residue at position 277 is Ala, Val,Ser, or Thr.

SEQ ID NO: 19 is the amino acid sequence of a Thermotoga sp. RQ2 acetylxylan esterase variant derived from“RQ2(a)” from U.S. Patent ApplicationPublication No. 2010-0087529, where the Xaa residue at position 277 isAla, Val, Ser, or Thr.

SEQ ID NO: 20 is the amino acid sequence of a Thermotoga sp. RQ2 acetylxylan esterase variant derived from “RQ2(b)” from U.S. PatentApplication Publication No. 2010-0087529, where the Xaa residue atposition 278 is Ala, Val, Ser, or Thr.

SEQ ID NO: 21 is the amino acid sequence of a Thermotoga lettingaeacetyl xylan esterase.

SEQ ID NO: 22 is the amino acid sequence of a Thermotoga petrophilaacetyl xylan esterase.

SEQ ID NO: 23 is the amino acid sequence of a first acetyl xylanesterase from Thermotoga sp. RQ2 described as “RQ2(a)”.

SEQ ID NO: 24 is the amino acid sequence of a second acetyl xylanesterase from Thermotoga sp. RQ2 described as “RQ2(b)”.

SEQ ID NO: 25 is the amino acid sequence of a Thermoanearobacteriumsaccharolyticum cephalosporin C deacetylase.

SEQ ID NO: 26 is the amino acid sequence of the acetyl xylan esterasefrom Lactococcus lactis (GENBANK® accession number ABX75634.1).

SEQ ID NO: 27 is the amino acid sequence of the acetyl xylan esterasefrom Mesorhizobium loti (GENBANK® accession number BAB53179.1).

SEQ ID NO: 28 is the amino acid sequence of the acetyl xylan esterasefrom Geobacillus stearothermophilus (GENBANK® accession numberAAF70202.1).

SEQ ID NOs 29-163 are the amino acid sequences of peptides havingaffinity to an oral cavity surface.

SEQ ID NOs: 164-177 are the amino acid sequences of peptidelinkers/spacers. SEQ ID NOs: 178-197 are the amino acid sequences ofvarious targeted perhydrolase fusion constructs comprising aperhydrolytic enzyme couple via a peptide linker to a binding domainhaving affinity for an oral surface (see International PatentApplication Publication No. WO2012/087970A2 to Butterick et al.).

DETAILED DESCRIPTION

The following description of the preferred embodiment(s) is merelyexemplary in nature and is in no way intended to limit the invention,its application, or uses.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an”, and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of” and “consisting of”. Similarly, the term“consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or use solutions in the realworld; through inadvertent error in these procedures; throughdifferences in the manufacture, source, or purity of the ingredientsemployed to make the compositions or carry out the methods; and thelike. The term “about” also encompasses amounts that differ due todifferent equilibrium conditions for a composition resulting from aparticular initial mixture. Whether or not modified by the term “about”,the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the terms “substrate”, “suitable substrate”, “acyldonor”, and “carboxylic acid ester substrate” interchangeably referspecifically to:

(a) one or more esters having the structure

[X]_(m)R₅

-   -   wherein    -   X is an ester group of the formula R₆C(O)O;    -   R₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with a hydroxyl group or C1 to C4 alkoxy        group, wherein R₆ optionally comprises one or more ether        linkages where R₆ is C2 to C7;    -   R₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety        or a cyclic five-membered heteroaromatic or six-membered cyclic        aromatic or heteroaromatic moiety optionally substituted with a        hydroxyl group; wherein each carbon atom in R₅ individually        comprises no more than one hydroxyl group or no more than one        ester group, and wherein R₅ optionally comprises one or more        ether linkages;    -   m is an integer ranging from 1 to the number of carbon atoms in        R₅,    -   said one or more esters having solubility in water of at least 5        ppm at 25° C.; or

(b) one or more glycerides having the structure

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O); or

(c) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; or

(d) one or more acetylated monosaccharides, acetylated disaccharides, oracetylated polysaccharides; or

(e) any combination of (a) through (d).

As used herein, the term “peracid” is synonymous with peroxyacid,peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoicacid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0.

As used herein, the term “monoacetin” is synonymous with glycerolmonoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term “diacetin” is synonymous with glyceroldiacetate; glycerin diacetate, glyceryl diacetate, and all othersynonyms of CAS Registry Number 25395-31-7.

As used herein, the term “triacetin” is synonymous with glycerintriacetate; glycerol triacetate; glyceryl triacetate,1,2,3-triacetoxypropane; 1,2,3-propanetriol triacetate and all othersynonyms of CAS Registry Number 102-76-1.

As used herein, the terms “acetylated sugar” and “acetylated saccharide”refer to mono-, di- and polysaccharides comprising at least one acetylgroup. Examples include, but are not limited to glucose pentaacetate;xylose tetraacetate; acetylated xylan; acetylated xylan fragments;β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; andtri-O-acetyl-glucal.

As used herein, the terms “hydrocarbyl”, “hydrocarbyl group”, and“hydrocarbyl moiety” is meant a straight chain, branched or cyclicarrangement of carbon atoms connected by single, double, or triplecarbon to carbon bonds and/or by ether linkages, and substitutedaccordingly with hydrogen atoms. Such hydrocarbyl groups may bealiphatic and/or aromatic. Examples of hydrocarbyl groups includemethyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, cyclopropyl,cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl, hexyl, cyclohexyl,benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety isa straight chain, branched or cyclic arrangement of carbon atomsconnected by single carbon to carbon bonds and/or by ether linkages, andsubstituted accordingly with hydrogen atoms.

As used herein, the terms “monoesters” and “diesters” of 1,2-ethanediol;1,2-propanediol; 1,3-propanediol; 1,2-butanediol; 1,3-butanediol;2,3-butanediol; 1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol;1,5-pentandiol; 1,6-pentanediol; 1,2-hexanediol; 2,5-hexanediol;1,6-hexanediol; and mixtures thereof, refer to said compounds comprisingat least one ester group of the formula RC(O)O, wherein R is a C1 to C7linear hydrocarbyl moiety. In one embodiment, the carboxylic acid estersubstrate is selected from the group consisting of propylene glycoldiacetate (PGDA), ethylene glycol diacetate (EDGA), and mixturesthereof.

As used herein, the term “propylene glycol diacetate” is synonymous with1,2-diacetoxypropane, propylene diacetate, 1,2-propanediol diacetate,and all other synonyms of CAS Registry Number 623-84-7.

As used herein, the term “ethylene glycol diacetate” is synonymous with1,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all othersynonyms of CAS Registry Number 111-55-7.

As used herein, the terms “suitable enzymatic reaction mixture”,“components suitable for in situ generation of a peracid”, “suitablereaction components”, “suitable aqueous reaction mixture”, “reactionmixture”, and “peracid-generating components” refer to the materials andwater (from saliva and/or applied by the user to the hydratable adhesivefilm prior to use) in which the reactants and the perhydrolytic enzymecatalyst come into contact. The peracid-generating components willinclude at least enzyme having perhydrolytic activity, preferablywherein the perhydrolytic enzyme is at least one CE-7 perhydrolase(optionally in the form of a fusion protein targeted to a body surface),at least one suitable carboxylic acid ester substrate, a source ofperoxygen, and water (from saliva and/or applied by the user to thehydratable adhesive film prior to use).

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate with peroxide to form a peracid. Typically, inorganicperoxide is reacted with the selected substrate in the presence of acatalyst to produce the peroxycarboxylic acid. As used herein, the term“chemical perhydrolysis” includes perhydrolysis reactions in which asubstrate (a peroxycarboxylic acid precursor) is combined with a sourceof hydrogen peroxide wherein peroxycarboxylic acid is formed in theabsence of an enzyme catalyst. As used herein, the term “enzymaticperhydrolysis” includes perhydrolysis reactions in which a carboxylicacid ester substrate (a peracid precursor; the “acyl donor”) is combinedwith a source of hydrogen peroxide and water whereby the enzyme catalystcatalyzes the formation of peracid.

As used herein, the term “perhydrolase activity” refers to the catalystactivity per unit mass (for example, milligram) of protein, dry cellweight, or immobilized catalyst weight.

As used herein, “one unit of enzyme activity” or “one unit of activity”or “U” is defined as the amount of perhydrolase activity required forthe production of 1 μmol of peroxycarboxylic acid product per minute ata specified temperature.

As used herein, the terms “enzyme catalyst” and “perhydrolase catalyst”refer to a catalyst comprising an enzyme having perhydrolysis activityand may be in the form of a whole microbial cell, permeabilizedmicrobial cell(s), one or more cell components of a microbial cellextract, partially purified enzyme, or purified enzyme. The enzymecatalyst may also be chemically modified (such as by pegylation or byreaction with cross-linking reagents). The perhydrolase catalyst mayalso be immobilized on a soluble or insoluble support using methodswell-known to those skilled in the art; see for example, Immobilizationof Enzymes and Cells; Gordon F. Bickerstaff, Editor; Humana Press,Totowa, N.J., USA; 1997. In one embodiment, the perhydrolase catalystmay be immobilized non-covalently in or on an oral care strip (e.g., awhitening strip) or dental tray. In a further embodiment, thenon-covalent immobilization to the strip or dental tray may be throughthe use of a peptidic binding domain having strong affinity for amaterial in or on the strip or tray (e.g., a fusion protein comprising aperhydrolytic enzyme coupled through an optional peptide spacer to apeptidic binding domain). In another embodiment, the dental tray isdeformable tray. In yet a further embodiment, the perhydrolase catalystis immobilized in or on the deformable tray after the formation of thedental impression.

As used herein, “acetyl xylan esterases” refers to an enzyme (E.C.3.1.1.72; AXEs) that catalyzes the deacetylation of acetylated xylansand other acetylated saccharides.

As used herein, the terms “cephalosporin C deacetylase” and“cephalosporin C acetyl hydrolase” refer to an enzyme (E.C. 3.1.1.41)that catalyzes the deacetylation of cephalosporins such as cephalosporinC and 7-aminocephalosporanic acid (Mitsushima et al., (1995) Appl. Env.Microbiol. 61(6):2224-2229). The amino acid sequences of severalcephalosporin C deacetylases having significant perhydrolytic activityare provided herein.

As used herein, the term “Bacillus subtilis ATCC® 31954™” refers to abacterial cell deposited to the American Type Culture Collection (ATCC)having international depository accession number ATCC® 31954™. Asdescribed herein, an enzyme having significant perhydrolase activityfrom B. subtilis ATCC® 31954™ is provided as SEQ ID NO: 4 (see UnitedStates Patent Application Publication No. 2010-0041752).

As used herein, the term “Thermotoga maritima MSB8” refers to abacterial cell reported to have acetyl xylan esterase activity (GENBANK®NP 227893.1; see U.S. Patent Application Publication No. 2008-0176299).The amino acid sequence of the enzyme having perhydrolase activity fromThermotoga maritima MSB8 is provided as SEQ ID NO: 11. Variants of theThermotoga maritima MSB8 perhydrolase are provided as SEQ ID NOs: 1 and16.

As used herein, an “isolated nucleic acid molecule”, “isolatedpolynucleotide”, and “isolated nucleic acid fragment” will be usedinterchangeably and refer to a polymer of RNA or DNA that is single- ordouble-stranded, optionally containing synthetic, non-natural or alterednucleotide bases. An isolated nucleic acid molecule in the form of apolymer of DNA may be comprised of one or more segments of cDNA, genomicDNA or synthetic DNA.

The term “amino acid” refers to the basic chemical structural unit of aprotein or polypeptide. The following abbreviations are used herein toidentify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any amino acid or asdefined herein Xaa X

As used herein, the term “about” modifying the quantity of an ingredientor reactant employed refers to variation in the numerical quantity thatcan occur, for example, through typical measuring and liquid handlingprocedures used for making concentrates or As used herein, the terms“signature motif” and “diagnostic motif” refer to conserved structuresshared among a family of enzymes having a defined activity. Thesignature motif can be used to define and/or identify the family ofstructurally-related enzymes having similar enzymatic activity for adefined family of substrates. The signature motif can be a singlecontiguous amino acid sequence or a collection of discontiguous,conserved motifs that together form the signature motif. Typically, theconserved motif(s) is represented by an amino acid sequence. In oneembodiment, the perhydrolytic enzymes used in the present compositionsand methods comprise a CE-7 carbohydrate esterase signature motif.

As used herein, the term “sequence analysis software” refers to anycomputer algorithm or software program that is useful for the analysisof nucleotide or amino acid sequences. “Sequence analysis software” maybe commercially available or independently developed. Typical sequenceanalysis software will include, but is not limited to, the GCG suite ofprograms (Wisconsin Package Version 9.0, Accelrys Software Corp., SanDiego, Calif.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St.Madison, Wis. 53715 USA), CLUSTALW (for example, version 1.83; Thompsonet al., Nucleic Acids Research, 22(22):4673-4680 (1994)), and the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York,N.Y.), Vector NTI (Informax, Bethesda, Md.) and Sequencher v. 4.05.Within the context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters set by the softwaremanufacturer that originally load with the software when firstinitialized.

The term “body surface” refers to any surface of the human body that mayserve as the target for a benefit agent, such as a peracid benefitagent. The present methods and compositions are directed to oral careapplications and products. As such, the body surface comprises an oralcavity material/surface. In one embodiment, the oral cavity materialcomprises tooth enamel.

As used herein, the terms “tooth whitening” and “tooth bleaching” areused interchangeably, to refer to improving the brightness (e.g.,whitening) of a tooth or teeth. Whitening strips are described hereincomprising ingredients suitable to enzymatically generate an efficaciousamount of a peracid to whiten teeth when hydrated.

As used in herein, “intrinsic stains” in teeth refer to the resultingcolor from chromogens within the enamel and underlying dentin. Theintrinsic color of human teeth tends to become more yellow with aging,due to the thinning of the enamel and darkening of the underlying yellowdentin. Removal of intrinsic stain usually requires the use of peroxidesor other oxidizing chemicals, which penetrate the enamel and decolorizethe internal chromogens.

In contrast to intrinsic stains, “extrinsic stains” form on the surfaceof the teeth when exogenous chromogenic materials bind to the enamel,usually within the pellicle naturally coating the teeth. Most peopleaccumulate some degree of unsightly extrinsic stains on their teeth overtime. This staining process is promoted by such factors as: (1) theingestion of tannin-containing foods and beverages such as coffee, tea,or red wine; (2) the use of tobacco products; and/or (3) exposure tocertain cationic substances (e.g., tin, iron, and chlorhexidine). Thesesubstances tend to adhere to the enamel's hydroxyapatite structure,which leads to tooth discoloration and a concomitant reduction in toothwhiteness. Over a period of years, extrinsic stains may penetrate theenamel layer and result in intrinsic stains.

As used herein, the term “destain” or “destaining” refers to the processof removing a stain from an oral cavity surface. The stain(s) may beintrinsic stains, extrinsic stains, or a combination thereof.

As used herein, “effective amount of perhydrolase enzyme” refers to thequantity of perhydrolase enzyme necessary to achieve the enzymaticactivity required in the specific application. Such effective amountsare readily ascertained by one of ordinary skill in the art and arebased on many factors, such as the particular enzyme variant used.

As used herein, the terms “peroxygen source” and “source of peroxygen”refer to compounds capable of providing hydrogen peroxide at aconcentration of about 1 mM or more when in an aqueous solutionincluding, but not limited to, hydrogen peroxide, hydrogen peroxideadducts (e.g., urea-hydrogen peroxide adduct (carbamide peroxide)),perborates, and percarbonates. As described herein, the peroxygen sourcein the present whitening strips is in the form of granular particles,wherein the user hydrates the granular peroxide particles to release aneffective amount of hydrogen peroxide. As described herein, theconcentration of the hydrogen peroxide provided by the peroxygencompound in the aqueous reaction formulation is initially at least 0.1mM or more upon combining the reaction components. In one embodiment,the hydrogen peroxide concentration in the aqueous reaction formulationis at least 0.5 mM. In one embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is at least 1 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction formulation is at least 10 mM. In another embodiment, thehydrogen peroxide concentration in the aqueous reaction formulation isat least 100 mM. In another embodiment, the hydrogen peroxideconcentration in the aqueous reaction formulation is at least 200 mM. Inanother embodiment, the hydrogen peroxide concentration in the aqueousreaction formulation is 500 mM or more. In yet another embodiment, thehydrogen peroxide concentration in the aqueous reaction formulation is1000 mM or more. The molar ratio of the hydrogen peroxide to enzymesubstrate, e.g., triglyceride, (H₂O₂:substrate) in the formulation maybe from about 0.002 to 20, preferably about 0.1 to 10, and mostpreferably about 0.5 to 5.

As used herein, the term “oligosaccharide” refers to compoundscontaining between 2 and at least 24 monosaccharide units linked byglycosidic linkages. The term “monosaccharide” refers to a compound ofempirical formula (CH₂O)_(n), where n>3, the carbon skeleton isunbranched, each carbon atom except one contains a hydroxyl group, andthe remaining carbon atom is an aldehyde or ketone at carbon atom 1. Theterm “monosaccharide” also refers to intracellular cyclic hemiacetal orhemiketal forms.

As used herein, the term “hydratable adhesive” will refer to an adhesivematerial capable of being hydrated. The hydratable adhesive issubstantially dry and non-adhesive until hydrated. Upon hydration, thehydratable adhesive becomes sufficiently adhesive to bind the toothwhitening strip/film to the surface of a tooth. The hydratable adhesivefilm also comprises a granular bleaching ingredient whereby uponhydration and effective amount of hydrogen peroxide is released to beused in the enzymatic formation of a peracid bleaching agent. Thewhitening strip/film is typically thin (typically less than 2 mm),shaped and sized to fit within the oral cavity, and sufficientlyflexible such that the film and be applied and placed in contact with aplurality of teeth whereby the hydrated adhesive helps to hold thefilm/strip on the tooth surface and provide a sufficient amount of timefor the peracid bleaching agent to whiten the teeth.

As used herein, the term “effective amount” will refer to the amount ofmaterial necessary to achieve the desired effect.

As used herein, the term “substantially non-adhesive until hydrated”will refer to the lack of adhesive strength sufficient to adhere thetooth whitening film to the surface of a plurality of teeth prior tohydration. As such, the hydratable adhesive film will be easy to handleand manipulate prior to application/hydration by the user.

By “sequence identity” is meant amino acid identity using a sequencealignment program, e.g., ClustalW or BLAST, e.g., generally as describedin Altschul S F, Gish W, Miller W, Myers E W, Lipman D J, “Basic localalignment search tool”, J Mol Biol (1990) 215 (3): 403-410, and GoujonM, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, Lopez R, NucleicAcids Research (2010) 38 Suppl: W695-9.

Acyl donors for use in the present invention, for example, to formperacids upon reaction with peroxide, are selected from one or more of(i) C₂₋₁₈ carboxylic acids, e.g C₂₋₆ carboxylic acids (e.g., aceticacid), including lower linear or branched alkyl carboxylic acids,optionally substituted with hydroxy and/or C₁₋₄ alkoxy; (ii)hydrolysable and acceptable esters thereof (e.g. mono-, di-, andtri-glycerides and acylated saccharides) and (iii) mixtures thereof. Forexample, acyl donors include 1,2,3-triacetoxypropane (sometimes referredto herein as triacetin or glycerin triacetate) and acylated saccharides,e.g. acetylated saccharides. In a particular embodiment, esters for thisuse may, for example, be esters having solubility in water of at least 5ppm at 25° C.

The acyl donors and/or enzymes may optionally be encapsulated. There area variety of encapsulation options well-known to the art, both naturaland synthetic. Modified starches and gum Arabic are particularlywell-suited since they are food grade, relatively inexpensive, quick todissolve, and can adsorb fairly high levels of liquid oils. Any impacton the final viscosity needs to be considered.

In some embodiments, the granules comprise an antisensitivity agentcapable of desensitizing the nerves or occluding dentine tubules. Insome embodiments, the antisensitivity agent is selected from a potassiumion source, a silicate, a stannous ion source, a basic amino acid, aclay, and a combination thereof. In some embodiments, the potassium ionsource is an orally-acceptable potassium salt and is present in anamount effective to reduce dentinal sensitivity. In some embodiments,the potassium ion source is selected from potassium chloride, potassiumnitrate and a combination thereof. In some embodiments, the basic aminoacid is arginine. In some embodiments, the basic amino acid is selectedfrom arginine phosphate, arginine bicarbonate, and argininehydrochloride. In some embodiments, the silicate is calcium silicate.

CE-7 Perhydrolases

The present compositions and method comprise enzymes havingperhydrolytic activity that are structurally classified as members ofthe carbohydrate family esterase family 7 (CE-7 family) of enzymes (seeCoutinho, P. M., Henrissat, B. “Carbohydrate-active enzymes: anintegrated database approach” in Recent Advances in CarbohydrateBioengineering, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3-12.). TheCE-7 family of enzymes has been demonstrated to be particularlyeffective for producing peroxycarboxylic acids from a variety ofcarboxylic acid ester substrates when combined with a source ofperoxygen (U.S. Pat. Nos. 7,794,378; 7,951,566; 7,723,083; and 7,964,378and U.S. Patent Application Publication Nos. 2008-0176299, 2010-0087529,2011-0081693, and 2011-0236335 to DiCosimo et al.; each incorporatedherein by reference).

Members of the CE-7 family include cephalosporin C deacetylases (CAHs;E.C. 3.1.1.41) and acetyl xylan esterases (AXEs; E.C. 3.1.1.72). Membersof the CE-7 esterase family share a conserved signature motif (Vincentet al., J. Mol. Biol., 330:593-606 (2003)). Perhydrolases comprising theCE-7 signature motif (“CE-7 perhydrolases”) and/or a substantiallysimilar structure are suitable for use in the compositions and methodsdescribed herein. Means to identify substantially similar biologicalmolecules are well known in the art (e.g., sequence alignment protocols,nucleic acid hybridizations and/or the presence of a conserved signaturemotif). In one aspect, the perhydrolase includes an enzyme comprisingthe CE-7 signature motif and at least 20%, preferably at least 30%, morepreferably at least 33%, more preferably at least 40%, more preferablyat least 42%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, morepreferably at least 90%, and most preferably at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to one of thesequences provided herein.

As used herein, the phrase “enzyme is structurally classified as a CE-7enzyme”, “CE-7 perhydrolase” or “structurally classified as acarbohydrate esterase family 7 enzyme” will be used to refer to enzymeshaving perhydrolytic activity which are structurally classified as aCE-7 carbohydrate esterase. This family of enzymes can be defined by thepresence of a signature motif (Vincent et al., supra). The signaturemotif for CE-7 esterases comprises three conserved motifs (residueposition numbering relative to reference sequence SEQ ID NO: 1; a C277Svariant of the Thermotoga maritima perhydrolase).

Arg118-Gly119-Gln120;

Gly186-Xaa187-Ser188-Gln189-Gly190; and

His303-Glu304.

Typically, the Xaa at amino acid residue position 187 is glycine,alanine, proline, tryptophan, or threonine. Two of the three amino acidresidues belonging to the catalytic triad are in bold. In oneembodiment, the Xaa at amino acid residue position 187 is selected fromthe group consisting of glycine, alanine, proline, tryptophan, andthreonine.

Further analysis of the conserved motifs within the CE-7 carbohydrateesterase family indicates the presence of an additional conserved motif(LXD at amino acid positions 272-274 of SEQ ID NO: 1) that may be usedto further define a perhydrolase belonging to the CE-7 carbohydrateesterase family. In a further embodiment, the signature motif definedabove may include an additional (fourth) conserved motif defined as:

Leu272-Xaa273-Asp274.

The Xaa at amino acid residue position 273 is typically isoleucine,valine, or methionine. The fourth motif includes the aspartic acidresidue (bold) belonging to the catalytic triad (Ser188-Asp274-His303).

The CE-7 perhydrolases may be in the form of fusion proteins having atleast one peptidic component having affinity for at least one bodysurface. In one embodiment, all alignments used to determine if atargeted perhydrolase (fusion protein) comprises the CE-7 signaturemotif will be based on the amino acid sequence of the perhydrolyticenzyme without the peptidic component having the affinity for a bodysurface.

A number of well-known global alignment algorithms (i.e., sequenceanalysis software) may be used to align two or more amino acid sequencesrepresenting enzymes having perhydrolase activity to determine if theenzyme is comprised of the present signature motif. The alignedsequence(s) are compared to the reference sequence (SEQ ID NO: 1) todetermine the existence of the signature motif. In one embodiment, aCLUSTAL alignment (such as CLUSTALW) using a reference amino acidsequence (as used herein the perhydrolase sequence (SEQ ID NO: 1)) isused to identify perhydrolases belonging to the CE-7 esterase family.The relative numbering of the conserved amino acid residues is based onthe residue numbering of the reference amino acid sequence to accountfor small insertions or deletions (for example, typically five aminoacids of less) within the aligned sequence.

Examples of other suitable algorithms that may be used to identifysequences comprising the present signature motif (when compared to thereference sequence) include, but are not limited to, Needleman andWunsch (J. Mol. Biol. 48, 443-453 (1970); a global alignment tool) andSmith-Waterman (J. Mol. Biol. 147:195-197 (1981); a local alignmenttool). In one embodiment, a Smith-Waterman alignment is implementedusing default parameters. An example of suitable default parametersinclude the use of a BLOSUM62 scoring matrix with GAP open penalty=10and a GAP extension penalty=0.5.

In one embodiment, suitable perhydrolases include enzymes comprising theCE-7 signature motif and at least 20%, preferably at least 30%, 33%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% amino acid identity to SEQ ID NO: 1.

Examples of suitable CE-7 carbohydrate esterases having perhydrolyticactivity include, but are not limited to, enzymes having an amino acidsequence such as SEQ ID NOs: 1, and 4-28. In one embodiment, the enzymecomprises an amino acid sequence selected from the group consisting of1, 10, 11, 15, and 16.

As used herein, the term “CE-7 variant”, “variant perhydrolase” or“variant” will refer to CE-7 perhydrolases having a genetic modificationthat results in at least one amino acid addition, deletion, and/orsubstitution when compared to the corresponding enzyme (typically thewild type enzyme) from which the variant was derived; so long as theCE-7 signature motif and the associated perhydrolytic activity aremaintained. CE-7 variant perhydrolases may also be used in the presentcompositions and methods. Examples of CE-7 variants are provided as SEQID NOs: 1, 15, 16, 17, 18, 19, and 20. In one embodiment, the variantsmay include SEQ ID NOs: 1 and 16.

The skilled artisan recognizes that substantially similar CE-7perhydrolase sequences (retaining the signature motifs) may also be usedin the present compositions and methods. In one embodiment,substantially similar sequences are defined by their ability tohybridize, under highly stringent conditions with the nucleic acidmolecules associated with sequences exemplified herein. In anotherembodiment, sequence alignment algorithms may be used to definesubstantially similar enzymes based on the percent identity to the DNAor amino acid sequences provided herein.

As used herein, a nucleic acid molecule is “hybridizable” to anothernucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when asingle strand of the first molecule can anneal to the other moleculeunder appropriate conditions of temperature and solution ionic strength.Hybridization and washing conditions are well known and exemplified inSambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor(2001). The conditions of temperature and ionic strength determine the“stringency” of the hybridization. Stringency conditions can be adjustedto screen for moderately similar molecules, such as homologous sequencesfrom distantly related organisms, to highly similar molecules, such asgenes that duplicate functional enzymes from closely related organisms.Post-hybridization washes typically determine stringency conditions. Oneset of preferred conditions uses a series of washes starting with 6×SSC,0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5%SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDSat 50° C. for 30 min. A more preferred set of conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringenthybridization conditions is 0.1×SSC, 0.1% SDS, 65° C. and washed with2×SSC, 0.1% SDS followed by a final wash of 0.1×SSC, 0.1% SDS, 65° C.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (Sambrook andRussell, supra). For hybridizations with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (Sambrookand Russell, supra). In one aspect, the length for a hybridizablenucleic acid is at least about 10 nucleotides. Preferably, a minimumlength for a hybridizable nucleic acid is at least about 15 nucleotidesin length, more preferably at least about 20 nucleotides in length, evenmore preferably at least 30 nucleotides in length, even more preferablyat least 300 nucleotides in length, and most preferably at least 800nucleotides in length. Furthermore, the skilled artisan will recognizethat the temperature and wash solution salt concentration may beadjusted as necessary according to factors such as length of the probe.

As used herein, the term “percent identity” is a relationship betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., andGriffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (von Heinje, G., ed.) Academic Press (1987); andSequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) StocktonPress, NY (1991). Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.), the AlignX program of Vector NTI v. 7.0 (Informax, Inc.,Bethesda, Md.), or the EMBOSS Open Software Suite (EMBL-EBI; Rice etal., Trends in Genetics 16, (6):276-277 (2000)). Multiple alignment ofthe sequences can be performed using the CLUSTAL method (such asCLUSTALW; for example version 1.83) of alignment (Higgins and Sharp,CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res.22:4673-4680 (1994); and Chenna et al., Nucleic Acids Res 31(13):3497-500 (2003)), available from the European Molecular BiologyLaboratory via the European Bioinformatics Institute) with the defaultparameters. Suitable parameters for CLUSTALW protein alignments includeGAP Existence penalty=15, GAP extension=0.2, matrix=Gonnet (e.g.,Gonnet250), protein ENDGAP=−1, protein GAPDIST=4, and KTUPLE=1. In oneembodiment, a fast or slow alignment is used with the default settingswhere a slow alignment is preferred. Alternatively, the parameters usingthe CLUSTALW method (e.g., version 1.83) may be modified to also useKTUPLE=1, GAP PENALTY=10, GAP extension=1, matrix=BLOSUM (e.g.,BLOSUM64), WINDOW=5, and TOP DIAGONALS SAVED=5.

In one aspect, suitable isolated nucleic acid molecules encode apolypeptide having an amino acid sequence that is at least about 20%,preferably at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequences reported herein. In another aspect, suitable isolated nucleicacid molecules encode a polypeptide having an amino acid sequence thatis at least about 20%, preferably at least 30%, 33%, 40%, 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the amino acid sequences reported herein. Suitable nucleic acidmolecules not only have the above homologies, but also typically encodea polypeptide having about 210 to 340 amino acids in length, about 300to about 340 amino acids, preferably about 310 to about 330 amino acids,and most preferably about 318 to about 325 amino acids in length whereineach polypeptide is characterized as having perhydrolytic activity.

Targeted Perhydrolases

As used herein, the term “targeted perhydrolase” and “targeted enzymehaving perhydrolytic activity” will refer to a fusion proteinscomprising at least one perhydrolytic enzyme (wild type or variantthereof) fused/coupled to at least one peptidic component havingaffinity for a target surface, preferably a targeted body surface. Theperhydrolytic enzyme within the targeted perhydrolase may be any CE-7carbohydrate esterase having perhydrolytic activity. The CE-7perhydrolase may be identified by the presence of the CE-7 signaturemotif that aligns with a reference sequence SEQ ID NO: 1, said signaturemotif comprising:

-   -   i) an RGQ motif at positions corresponding to positions 118-120        of SEQ ID NO: 1;    -   ii) a GXSQG motif at positions corresponding to positions        186-190 of SEQ ID NO:1; and    -   iii) an HE motif at positions corresponding to positions 303-304        of SEQ ID NO:1; and

In one embodiment, perhydrolytic enzymes may be those having an aminoacid sequence that is at least about 20%, preferably at least 30%, 33%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to any of the amino acid sequences reported herein(i.e., SEQ ID NOs 1, and 4-28).

In another embodiment, the fusion protein comprises a perhydrolyticenzyme having an amino acid sequence selected from the group consistingof SEQ ID NOs: 1, and 4-28.

As used herein the terms “peptidic component”, “peptidic componenthaving affinity for an oral cavity surface”, “oral cavity bindingdomain”, and “OCBD” will refer to component of the fusion protein thatis not part of the perhydrolytic enzyme comprising at least one polymerof two or more amino acids joined by a peptide bond; wherein thecomponent has affinity for the target oral cavity surface. In apreferred aspect, the OCBD has affinity for tooth enamel.

In one embodiment, the peptidic component having affinity for a bodysurface may be an antibody, an Fab antibody fragment, a single chainvariable fragment (scFv) antibody, a Camelidae antibody (Muyldermans,S., Rev. Mol. Biotechnol. (2001) 74:277-302), a non-antibody scaffolddisplay protein (Hosse et al., Prot. Sci. (2006) 15(1): 14-27 and Binz,H. et al. (2005) Nature Biotechnology 23, 1257-1268 for a review ofvarious scaffold-assisted approaches) or a single chain polypeptidelacking an immunoglobulin fold. In another aspect, the peptidiccomponent having affinity for the oral cavity tissue/surface (such astooth enamel) is a single chain peptide lacking an immunoglobulin fold.

The peptidic component having affinity for an oral cavity surface may beseparated from the perhydrolytic enzyme by an optional peptide linker.Certain peptide linkers/spacers are from 1 to 100 or 1 to 50 amino acidsin length. In some embodiments, the peptide spacers are about 1 to about25, 3 to about 40, or 3 to about 30 amino acids in length. In otherembodiments are spacers that are about 5 to about 20 amino acids inlength. Multiple peptide linkers may be used. In one embodiment, atleast one peptide linker is present and may be repeated up to 10 times.

In one embodiment, the fusion peptide comprises at least one oral cavitysurface-binding peptide selected from the group consisting of SEQ IDNOs: 178-197.

In another embodiment the target surface is a material that is part ofthe packaging, such as the whitening strip or polymeric backing layer(when using a polymeric backing layer to which the hydratable adhesiveis applied) and/or method of delivery to the oral cavity. The peptidiccomponent is selected for it affinity to a material or materials in usesuch as polymers, plastics and films. The targeted perhydrolase fusionprotein design allows for the controlled delivery and removal of theperhydrolase from the user by maintaining it on a removable device suchas, but not limited to, a mouth tray or strip.

The peptidic component having affinity for an oral cavity surface may beseparated from the CE-7 perhydrolase by an optional peptide linker.Certain peptide linkers/spacers are from 1 to 100 or 1 to 50 amino acidsin length. In some embodiments, the peptide spacers are about 1 to about25, 3 to about 40, or 3 to about 30 amino acids in length. In otherembodiments are spacers that are about 5 to about 20 amino acids inlength. Multiple peptide linkers may be used. Examples of peptidelinkers are provided as SEQ ID NOs: 164-177.

As such, examples of targeted CE-7 perhydrolases may include, but arenot limited to, any of the CE-7 perhydrolases having an amino acidsequence selected from the group consisting of SEQ ID NOs 1, and 4-28coupled to a peptidic component having affinity for an oral cavitysurface. In a preferred embodiment, examples of targeted perhydrolasesmay include, but are not limited to, any of CE-7 perhydrolases having anamino acid sequence selected from the group consisting of SEQ ID NOs 1,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, and 28 coupled to one or more body surface-bindingpeptides having affinity for an oral cavity surface (optionally througha peptide spacer). In a preferred embodiment, the targeted perhydrolaseincludes a CE-7 perhydrolase having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 1 and 16.

In one embodiment, the perhydrolase is a CE-7 perhydrolase in the formof a fusion protein having the following general structure:

PAH-[L]y-OCBD or OCBD-[L]y-PAH

wherein PAH is the enzyme having perhydrolytic activity, e.g., having aCE-7 signature motif, e.g., SEQ ID NO:1, and OCBD is a peptidiccomponent having affinity for an oral cavity surface; and L is anoptional linker; and y is an integer ranging from 0 to 10. In oneembodiment, the linker (L) is present and is a peptide linker rangingfrom 1 to 100 amino acids in length.

For example SEQ ID NO: 2 is a fusion protein having a perhydrolasesequence of SEQ ID NO: 1 coupled to a C-terminal targeting domain withan affinity for oral tissues.

The perhydrolases for use in the products and methods of the inventionmay be in free, protected (e.g., acetylated), or salt form.

In another embodiment, the target surface is a material that is part ofthe packaging and or delivery to the oral cavity. The peptidic componentis selected for it affinity to a material or materials in use such aspolymers, plastics and films. The targeted CE-7 perhydrolase fusionprotein design allows for the controlled delivery and removal of theperhydrolase from the user by maintaining it on a removable device suchas a mouth tray or strip.

Binding Affinity

The peptidic component having affinity for the oral cavity surfacecomprises a binding affinity for an oral cavity surface of 10⁻⁵ molar(M) or less. In certain embodiments, the peptidic component is one ormore oral cavity surface-binding peptides and/or binding domain(s)having a binding affinity of 10⁻⁵ molar (M) or less for tooth enamel. Insome embodiments, the binding peptides or domains will have a bindingaffinity value of 10⁻⁵ M or less in the presence of at least about50-500 mM salt. The term “binding affinity” refers to the strength ofthe interaction of a binding peptide with its respective substrate.Binding affinity can be defined or measured in terms of the bindingpeptide's dissociation constant (“K_(D)”), or “MB₅₀.”

“K_(D)” corresponds to the concentration of peptide at which the bindingsite on the target is half occupied, i.e., when the concentration oftarget with peptide bound (bound target material) equals theconcentration of target with no peptide bound. The smaller thedissociation constant, the more tightly the peptide is bound. Forexample, a peptide with a nanomolar (nM) dissociation constant bindsmore tightly than a peptide with a micromolar (04) dissociationconstant. Certain embodiments of the invention will have a K_(D) valueof 10⁻⁵ or less.

“MB₅₀” refers to the concentration of the binding peptide that gives asignal that is 50% of the maximum signal obtained in an ELISA-basedbinding assay. See, e.g., Example 3 of U.S. Patent ApplicationPublication 2005/022683; hereby incorporated by reference. The MB₅₀provides an indication of the strength of the binding interaction oraffinity of the components of the complex. The lower the value of MB₅₀,the stronger, i.e., “better,” the interaction of the peptide with itscorresponding substrate. For example, a peptide with a nanomolar (nM)MB₅₀ binds more tightly than a peptide with a micromolar (μM) MB₅₀.Certain embodiments of the invention will have a MB₅₀ value of 10⁻⁵ M orless.

In some embodiments, the peptidic component having affinity for an oralcavity surface may have a binding affinity, as measured by K_(D) or MB₅₀values, of less than or equal to about 10⁻⁵ M, less than or equal toabout 10⁻⁶ M, less than or equal to about 10⁻⁷ M, less than or equal toabout 10⁻⁸ M, less than or equal to about 10⁻⁹ M, or less than or equalto about 10⁻¹⁰ M.

In some embodiments, the oral cavity surface-binding peptides and/ororal cavity surface-binding domains may have a binding affinity, asmeasured by K_(D) or MB₅₀ values, of less than or equal to about 10⁻⁵ M,less than or equal to about 10⁻⁶ M, less than or equal to about 10⁻⁷ M,less than or equal to about 10⁻⁸ M, less than or equal to about 10⁻⁹ M,or less than or equal to about 10⁻¹⁰ M.

As used herein, the term “strong affinity” will refer to a bindingaffinity having a K_(D) or MB₅₀ value of less than or equal to about10⁻⁵ M, preferably less than or equal to about 10⁻⁶ M, more preferablyless than or equal to about 10⁻⁷ M, more preferably less than or equalto about 10⁻⁸ M, less than or equal to about 10⁻⁹ M, or most preferablyless than or equal to about 10⁻¹⁰ M.

Enzyme Powders

In some embodiments, the personal care compositions may use an enzymecatalyst in form of a stabilized enzyme powder. Methods to make andstabilize formulations comprising an enzyme powder are described in U.S.Patent Application Publication Nos. 2010-0086534 and 2010-0086535.

In one embodiment, the enzyme may be in the enzyme powder in an amountin a range of from about 0.5 weight percent (wt %) to about 75 wt %,e.g., 1 wt % to about 60 wt %, based on the dry weight of the enzymepowder. A preferred weight percent range of the enzyme in the enzymepowder/spray-dried mixture is from about 10 wt % to 50 wt %, and a morepreferred weight percent range of the enzyme in the enzymepowder/spray-dried mixture is from about 20 wt % to 33 wt %.

In one embodiment, the enzyme powder may further comprise an excipient.In one aspect, the excipient is provided in an amount in a range of fromabout 95 wt % to about 25 wt % based on the dry weight of the enzymepowder. A preferred wt % range of excipient in the enzyme powder is fromabout 90 wt % to 50 wt %, and a more preferred wt % range of excipientin the enzyme powder is from about 80 wt % to 67 wt %.

In one embodiment, the excipient used to prepare an enzyme powder may bean oligosaccharide excipient. In one embodiment, the oligosaccharideexcipient has a number average molecular weight of at least about 1250and a weight average molecular weight of at least about 9000. In someembodiments, the oligosaccharide excipient has a number averagemolecular weight of at least about 1700 and a weight average molecularweight of at least about 15000. Specific oligosaccharides may include,but are not limited to, maltodextrin, xylan, mannan, fucoidan,galactomannan, chitosan, raffinose, stachyose, pectin, insulin, levan,graminan, amylopectin, sucrose, lactulose, lactose, maltose, trehalose,cellobiose, nigerotriose, maltotriose, melezitose, maltotriulose,raffinose, kestose, and mixtures thereof. In a preferred embodiment, theoligosaccharide excipient is maltodextrin. Oligosaccharide-basedexcipients may also include, but are not limited to, water-solublenon-ionic cellulose ethers, such as hydroxymethyl-cellulose andhydroxypropylmethylcellulose, and mixtures thereof. In yet a furtherembodiment, the excipient may be selected from, but not limited to, oneor more of the following compounds: trehalose, lactose, sucrose,mannitol, sorbitol, glucose, cellobiose, α-cyclodextrin, andcarboxymethylcellulose.

Suitable Ester Substrates/Acyl Donors

Suitable carboxylic acid ester substrates may include esters having thefollowing formula:

-   -   (a) one or more esters having the structure

[X]_(m)R₅

-   -   wherein    -   X is an ester group of the formula R₆C(O)O;    -   R₆ is a C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with a hydroxyl group or C1 to C4 alkoxy        group, wherein R₆ optionally comprises one or more ether        linkages where R₆ is C2 to C7;    -   R₅ is a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety        or a five-membered cyclic heteroaromatic moiety or six-membered        cyclic aromatic or heteroaromatic moiety optionally substituted        with a hydroxyl group; wherein each carbon atom in R₅        individually comprises no more than one hydroxyl group or no        more than one ester group or carboxylic acid group, and wherein        R₅ optionally comprises one or more ether linkages;    -   m is an integer ranging from 1 to the number of carbon atoms in        R₅,    -   said one or more esters having solubility in water of at least 5        ppm at 25° C.; or    -   (b) one or more glycerides having the structure

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O); or    -   (c) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; or    -   (d) one or more acetylated monosaccharides, acetylated        disaccharides, or acetylated polysaccharides; or    -   (e) any combination of (a) through (d).

Suitable substrates may also include one or more acylated saccharidesselected from the group consisting of acylated mono-, di-, andpolysaccharides. In another embodiment, the acylated saccharides areselected from the group consisting of acetylated xylan; fragments ofacetylated xylan; acetylated xylose (such as xylose tetraacetate);acetylated glucose (such as α-D-glucose pentaacetate; β-D-glucosepentaacetate; 1-thio-β-D-glucose-2,3,4,6-tetraacetate); β-D-galactosepentaacetate; sorbitol hexaacetate; sucrose octaacetate;β-D-ribofuranose-1,2,3,5-tetraacetate;β-D-ribofuranose-1,2,3,4-tetraacetate; tri-O-acetyl-D-galactal;tri-O-acetyl-D-glucal; β-D-xylofuranose tetraacetate, β-D-glucopyranosepentaacetate; β-D-glucopyranose-1,2,3,4-tetraacetate;β-D-glucopyranose-2,3,4,6-tetraacetate;2-acetamido-2-deoxy-1,3,4,6-tetracetyl-β-D-glucopyranose;2-acetamido-2-deoxy-3,4,6-triacetyl-1-chloride-α-D-glucopyranose;β-D-mannopyranose pentaacetate, and acetylated cellulose. In a preferredembodiment, the acetylated saccharide is selected from the groupconsisting of β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; sucrose octaacetate; andacetylated cellulose.

In another embodiment, additional suitable substrates may also include5-acetoxymethyl-2-furaldehyde; 3,4-diacetoxy-1-butene; 4-acetoxybenezoicacid; vanillin acetate; propylene glycol methyl ether acetate; methyllactate; ethyl lactate; methyl glycolate; ethyl glycolate; methylmethoxyacetate; ethyl methoxyacetate; methyl 3-hydroxybutyrate; ethyl3-hydroxybutyrate; and triethyl 2-acetyl citrate.

In another embodiment, suitable substrates are selected from the groupconsisting of: monoacetin; diacetin; triacetin; monopropionin;dipropionin; tripropionin; monobutyrin; dibutyrin; tributyrin; glucosepentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylanfragments; β-D-ribofuranose-1,2,3,5-tetraacetate;tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diestersof 1,2-ethanediol; 1,2-propanediol; 1,3-propanediol; 1,2-butanediol;1,3-butanediol; 2,3-butanediol; 1,4-butanediol; 1,2-pentanediol;2,5-pentanediol; 1,5-pentanediol; 1,6-pentanediol; 1,2-hexanediol;2,5-hexanediol; 1,6-hexanediol; and mixtures thereof. In anotherembodiment, the substrate is a C1 to C6 polyol comprising one or moreester groups. In a preferred embodiment, one or more of the hydroxylgroups on the C1 to C6 polyol are substituted with one or more acetoxygroups (such as 1,3-propanediol diacetate; 1,2-propanediol diacetate;1,4-butanediol diacetate; 1,5-pentanediol diacetate, etc.). In a furtherembodiment, the substrate is propylene glycol diacetate (PGDA), ethyleneglycol diacetate (EGDA), or a mixture thereof.

In a further embodiment, suitable substrates are selected from the groupconsisting of monoacetin, diacetin, triacetin, monopropionin,dipropionin, tripropionin, monobutyrin, dibutyrin, and tributyrin. Inyet another aspect, the substrate is selected from the group consistingof diacetin and triacetin. In a most preferred embodiment, the suitablesubstrate comprises triacetin.

The carboxylic acid ester is present at a concentration sufficient toproduce the desired concentration of peroxycarboxylic acid uponenzyme-catalyzed perhydrolysis. The carboxylic acid ester need not becompletely soluble in the reaction formulation, but has sufficientsolubility to permit conversion of the ester by the perhydrolasecatalyst to the corresponding peroxycarboxylic acid. The carboxylic acidester is present in the reaction formulation at a concentration of 0.05wt % to 40 wt % of the reaction formulation, preferably at aconcentration of 0.1 wt % to 20 wt % of the reaction formulation, andmore preferably at a concentration of 0.5 wt % to 10 wt % of thereaction formulation.

The peroxygen source is provided as granules deposited in or on thehydratable adhesive film and may include hydrogen peroxide adducts(e.g., urea-hydrogen peroxide adduct (carbamide peroxide)) perboratesalts, percarbonate salts and peroxide salts. The concentration ofperoxygen compound in the reaction formulation may range from 0.0033 wt% to about 50 wt %, preferably from 0.033 wt % to about 40 wt %, morepreferably from 0.1 wt % to about 30 wt %.

Many perhydrolase catalysts (whole cells, permeabilized whole cells, andpartially purified whole cell extracts) have been reported to havecatalase activity (EC 1.11.1.6). Catalases catalyze the conversion ofhydrogen peroxide into oxygen and water. In one aspect, theperhydrolysis catalyst lacks catalase activity. In another aspect, acatalase inhibitor may be added to the reaction formulation. One ofskill in the art can adjust the concentration of catalase inhibitor asneeded. The concentration of the catalase inhibitor typically rangesfrom 0.1 mM to about 1 M; preferably about 1 mM to about 50 mM; morepreferably from about 1 mM to about 20 mM.

In another embodiment, the enzyme catalyst lacks significant catalaseactivity or may be engineered to decrease or eliminate catalaseactivity. The catalase activity in a host cell can be down-regulated oreliminated by disrupting expression of the gene(s) responsible for thecatalase activity using well known techniques including, but not limitedto, transposon mutagenesis, RNA antisense expression, targetedmutagenesis, and random mutagenesis.

The concentration of peroxycarboxylic acid generated (e.g. peraceticacid) by the perhydrolysis of at least one carboxylic acid ester is atleast about 0.1 ppm, preferably at least 0.5 ppm, 1 ppm, 5 ppm, 10 ppm,20 ppm, 100 ppm, 200 ppm, 300 ppm, 500 ppm, 700 ppm, 1000 ppm, 2000 ppm,5000 ppm or 10,000 ppm of peracid within 10 minutes, preferably within 5minutes, of initiating the perhydrolysis reaction. Clearly one of skillin the art can adjust the reaction components to achieve the desiredperacid concentration.

In one aspect, the reaction time required to produce the desiredconcentration of peracid is not greater than about two hours, preferablynot greater than about 30 minutes, more preferably not greater thanabout 10 minutes, and most preferably in about 5 minutes or less. Inother aspects, an oral cavity surface is contacted with theperoxycarboxylic acid formed in accordance with the processes describedherein within 5 minutes of hydrating and combining the reactioncomponents. In one embodiment, the tooth enamel is contacted with theperoxycarboxylic acid produced with the processes and compositionsdescribed herein within about 5 minutes to about 24 hours or withinabout 5 minutes to 2 hours of combining (via user hydration) saidreaction components present in or on the whitening strip/film.

HPLC Assay Method for Determining the Concentration of PeroxycarboxylicAcid and Hydrogen Peroxide

A variety of analytical methods can be used to analyze the reactants andproducts including, but not limited to, titration, high performanceliquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy(MS), capillary electrophoresis (CE), the analytical procedure describedby U. Pinkernell et al., (Anal. Chem., 69(17):3623-3627 (1997)), and the2,2′-azino-bis β-ethylbenzothazoline)-6-sulfonate (ABTS) assay (U.Pinkernell et. al. Analyst, 122: 567-571 (1997) and Dinu et. al. Adv.Funct. Mater., 20: 392-398 (2010)) as described in the present examples.

EXEMPLARY EMBODIMENTS Embodiment 1

A tooth whitening strip (Strip 1) comprising a hydratable adhesive filmwith a first side and a second side, the first side having a granularbleaching ingredient attached thereto,

wherein the strip comprises, in or on the film or in granules attachedto the first side of the film,

(i) a protein having perhydrolytic activity which contains the signaturemotif of a member of the carbohydrate esterase family 7;

(ii) an acyl donor, e.g., selected from carboxylic acid esters and acylcompounds,

Embodiment 2

Strip 1 wherein the protein having perhydrolase activity comprises anamino acid sequence selected from:

(SEQ ID NO: 1) a) MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNISPPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKG,

b) an amino acid sequence having i) an RGQ motif at positionscorresponding to positions 118-120 of SEQ ID NO: 1; ii) a GXSQG motif atpositions corresponding to positions 186-190 of SEQ ID NO: 1; and iii)an HE motif at positions corresponding to positions 303-304 of SEQ IDNO: 1; and

c) an amino acid sequence having at least 80% sequence identity to SEQID NO: 1.

Embodiment 3

Embodiment 1 or 2 wherein the protein having perhydrolytic activitycomprises an amino acid sequence which has affinity to, e.g., binds toor complexes with oral surfaces or alternatively has affinity, e.g.,binds to or complexes with, one or more components of the whiteningstrip.

Embodiment 4

Any of the foregoing strips comprising a protein having perhydrolaseactivity which binds to or complexes with oral surfaces (e.g., toothpellicle or enamel) comprising an amino acid sequence selected from

(SEQ ID NO: 2) a) MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNISPPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKGGPGSGGAGSPGSAGGPGSTKPPRTPTANTSRPHHNFGSGGGGSPHHHHHH, and

b) an amino acid sequence having at least 80% sequence identity to SEQID NO: 2.

Embodiment 5

Any of the foregoing strips wherein the protein having perhydrolyticactivity is provided in granular form on the first surface of the film.

Embodiment 6

Any of the foregoing strips wherein the second side of the hydratableadhesive film is attached to a layer which inhibits dissolution of thehydratable adhesive film.

Embodiment 7

Any of the foregoing strips wherein the granular bleaching ingredient iscoated with a quickly dissolving material, such as cornstarch, sodiumsulfate gum arabic, and combinations thereof.

Embodiment 8

Any of the foregoing strips wherein the granular bleaching ingredient isselected from granules comprising organic and/or inorganic oxidizers,e.g., selected from hydrogen peroxide, urea peroxide, percarbonates,perborates, peroxymonophosphates, peroxydisulfates, peroxyacids, andperacetic acid.

Embodiment 9

Any of the foregoing strips wherein the granular bleaching ingredient isselected from solid peroxides and solid peroxide donors, e.g., selectedfrom peroxide salts or complexes (e.g., such as peroxyphosphate,peroxycarbonate, perborate, peroxysilicate, or persulphate salts; forexample calcium peroxyphosphate, sodium perborate, sodium carbonateperoxide, sodium peroxyphosphate, and potassium persulfate);hypochlorites; urea peroxide; hydrogen peroxide polymer complexes suchas hydrogen peroxide-polyvinyl pyrrolidone polymer complexes; metalperoxides e.g. zinc peroxide and calcium peroxide; peracids, andcombinations thereof.

Embodiment 10

Any of the foregoing strips wherein the granular bleaching ingredientcomprises urea peroxide, a hydrogen peroxide-polyvinyl pyrrolidonepolymer complex, sodium percarbonate, or a combination of two or morethereof

Embodiment 11

Any of the foregoing strips where the particle size (D₅₀) of thegranules on the first surface of the film, e.g., the granular bleachingingredient, or perhydrolase or acyl donor in granular form, is 0.1-300microns, e.g. 10-275 microns, e.g., 100-250 microns.

Embodiment 12

Any of the foregoing strips wherein the granular bleaching ingredientcomprises greater than 0.01%, e.g. 0.01-0.1%, e.g. 0.02-0.08%, of thetotal weight of the hydratable adhesive film and a granular bleachingingredient attached thereto.

Embodiment 13

Any of the foregoing strips wherein the amount of granular bleachingagent on the first side of the hydratable adhesive film is 0.001-10mg/cm², e.g., 0.001-1 mg/cm², for example 0.005-0.015 mg/cm².

Embodiment 14

Any of the foregoing strips wherein the acyl donor is selected from (i)one or more C₂₋₁₈ carboxylic acid esters, e.g C₂₋₆ carboxylic acidesters (e.g., acetyl esters), including lower linear or branched alkylcarboxylic acid esters, optionally substituted with hydroxy and/or C₁₋₄alkoxy; (ii) one or more acylated glycerides (e.g. mono-, di-, andtri-glycerides), (iii) acylated saccharides, and (iv) mixtures thereof.

Embodiment 15

Any of the foregoing strips the acyldonor is selected from1,2,3-triacetoxypropane (sometimes referred to herein as triacetin orglycerin triacetate) and acylated saccharides, e.g. acylatedsaccharides.

Embodiment 16

Any of the foregoing strips comprising an acyl donor which comprises anester compound having solubility in water of at least 5 ppm at 25° C.

Embodiment 17

Any of the foregoing strips which comprises a peracid or which generatesa peracid upon use.

Embodiment 18

Any of the foregoing strips wherein the ingredients are present inamounts sufficient to provide, upon mixing, a bleaching agent in anamount and concentration effective to whiten teeth.

Embodiment 19

Any of the foregoing strips wherein the hydratable adhesive filmcomprises one or more water-soluble polymers selected from hydrophiliccellulose ethers (e.g. carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methylcellulose,), polyvinyl acetates, carbomers (e.g.,CARBOPOL® 971P), polysaccharide gums (e.g. xanthan gum), modified foodstarches, gelatin (e.g. animal or fish-based gelatin), cross-linkedcarboxyvinyl copolymers, cross-linked polyvinylpyrrolidones,polyethylene oxide (e.g, POLYOX™) polyacrylic acids and polyacrylates,polyvinyl alcohols, alginate, casein, pullulan, and combinationsthereof.

Embodiment 20

Any of the foregoing strips wherein the hydratable adhesive filmcomprises one or more water-soluble polymers selected from hydrophiliccellulose ethers (e.g. hydroxypropylmethyl cellulose or hydroxypropylcellulose), polyethylene oxide, polyvinyl acetates, and carbomers (e.g.,CARBOPOL® 971P); and combinations thereof

Embodiment 21

Any of the foregoing strips wherein the hydratable adhesive filmcomprises hydroxypropylmethyl cellulose, polyvinyl acetates, and acarbomer, for example, in a dry weight ratio of 10-20 HPMC:2-10 PVAc:1carbomer.

Embodiment 22

Any of the foregoing strips wherein the hydratable adhesive film furthercomprises a plasticizer, e.g. propylene glycol, polyethylene glycol ortriacetin.

Embodiment 23

Any of the foregoing strips wherein the first side of the hydratableadhesive film is covered by a protective cover prior to use.

Embodiment 24

Any of the foregoing strips wherein the hydratable adhesive film has aviscosity of at least 100,000 cps upon activation, e.g., viscosity of100,000 to 200,000 cps.

Embodiment 25

Any of the foregoing strips wherein the hydratable adhesive film issubstantially dry prior to application.

Embodiment 26

Any of the foregoing strips wherein the thickness of the hydratableadhesive film is 0.1-5 mm, e.g., 0.5-5 mm.

Embodiment 27

Any of the foregoing strips wherein the approximate overall dimensionsare 2-10 cm long×0.5-2 cm wide×0.1-10 mm thick, e.g., 1-10 mm thick, forexample a strip wherein the surface area of one side is 5-20 cm², e.g.,about 5-15 cm², e.g., about 10 cm².

Embodiment 28

Any of the foregoing strips comprising coated granules of a hydrogenperoxide-polyvinyl pyrrolidone polymer complex, urea peroxide and/orsodium percarbonate and granules of perhydrolase on the first surface ofthe film, with triacetin dispersed in the film.

Embodiment 29

Any of the foregoing strips, further comprising granules of anantisensitivity agent, e.g. potassium nitrate or arginine.

Embodiment 30

A method (Method 2) of whitening teeth comprising applying the firstside of a strip as hereinbefore described, e.g. Strip 1 et seq. directlyto the teeth, and leaving it on for a sufficient time, e.g., at least 5minutes, for example 10-60 minutes, e.g., 10-30 minutes, to whiten theteeth.

Embodiment 31

A method (Method 3) of making a strip for tooth whitening, e.g., a stripas hereinbefore described, according to Strip 1 et seq., comprisingproviding a semi-dry hydratable adhesive film, e.g., as hereinbeforedescribed, e.g., which film has been cast from water and not fullydried, or which film has been moistened, adding to one surface of thefilm granules of a granular bleaching ingredient, e.g., as hereinbeforedescribed, and drying the film with the granules added to one surface.

For example, the strips may be made by first making the hydratableadhesive film, using conventional means and then adding the granulatedwhitening ingredient to one surface. The hydratable adhesive film stripscan be cast from water in a variety of ways known in the art, such as byextrusion, or by casting from a water suspension (for example at asolids level of 10-30%) onto a heated belt, from which the water isevaporated. Alternatively, the film is dried, but then remoistened. Thegranules can be added to the surface of this film while the film issemi-dry, i.e. just moist enough to be tacky, so that the granules stickto the surface of the film. Once the film is fully dry and cooled toroom temperature, the granules continue to adhere to the surface of thefilm. Prior to use, therefore, the hydratable adhesive film and thestrip as a whole are substantially dry. Because the peroxide is on thesurface of the film only, a relatively small quantity of granules arerequired to provide an effective concentration at the surface.

PREFERRED EMBODIMENTS

When exposed to saliva or other sources of water (such as tap water),the granules dissolve and release the reaction components toenzymatically produce the desired peracid. Hydration of the hydratableadhesive layer increases the tackiness of the film, enabling thewhitening film/strip to bind to the target surface (i.e., tooth enamel).

The hydratable adhesive film comprises one or more water soluble, orallyacceptable polymers selected from hydrophilic cellulose ethers (e.g.,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose,), polyvinyl acetates, carbomers (e.g., CARBOPOL® 971P),polysaccharide gums (e.g., xanthan gum), modified food starches, gelatin(e.g., animal or fish-based gelatin), cross-linked carboxyvinylcopolymers, cross-linked polyvinylpyrrolidones, polyethylene oxide(e.g., POLYOX™), polyacrylic acids and polyacrylates, polyvinylalcohols, alginate, casein, pullulan, and combinations thereof. Adhesivegel formulations for use with tooth whitening agents are known in theart, for example, as described in U.S. Pat. Nos. 7,862,801; 5,746,598;6,730,316; and 7,128,899; each incorporated by reference in itsentirety. The adhesive film allows the peracid bleaching agent to stayin contact with the teeth for extended periods of time and protects softtissues, and thus should provide a high viscosity, for example, aviscosity upon application of at least 100,000 centipoise (cps) (about100 Pascal-second (Pa·s)), preferable 100,000 to 200,000 cps (100 to 200Pa·s).

Where a second film layer is used to protect the hydratable adhesivefilm from rapid degradation or dissolution, the carrier or backingmaterial may be made from textiles, cloth, wood composite, resin,elastomer, paper, insoluble or less soluble cellulose derivatives suchas ethyl cellulose and cellulose acetate, polyvinyl chloride, wax,PARAFILM™, polyethylene, polyvinyl alcohol, TEFLON™, polyvinyl chloride,polyvinyl acetate and their derivatives.

The granular bleaching ingredient may be a solid peroxide or solidperoxide donor selected from peroxide salts or complexes (such asperoxyphosphate, peroxycarbonate, perborate, peroxysilicate, orpersulphate salts; for example calcium peroxyphosphate, sodiumperborate, sodium carbonate peroxide, sodium peroxyphosphate, andpotassium persulfate), hypochlorites; urea peroxide; hydrogen peroxidepolymer complexes such as hydrogen peroxide-polyvinyl pyrrolidonepolymer complexes, and metal peroxides, for example, zinc peroxide andcalcium peroxide; a solid peracid; and combinations thereof. Inparticular embodiments, the granular bleaching ingredient is ureaperoxide or a hydrogen peroxide polyvinylpyrrolidone polymer complex.The granular bleaching ingredient may be optionally coated to provideimproved storage stability (for example, coated with sodium sulfate,corn starch or gum arabic).

Listing of the Preferred Embodiments Preferred Embodiment 1

A tooth whitening strip comprising a hydratable adhesive film with afirst side and a second side, the first side having a granular bleachingingredient attached thereto, wherein the tooth whitening strip furthercomprises, in or on the film or in the form of granules attached to thefirst side of the film;

a) an enzyme having perhydrolytic activity, said enzyme having acarbohydrate esterase family 7 (CE-7) signature motif that aligns with areference sequence SEQ ID NO: 1, said signature motif comprising:

-   -   i) an RGQ motif at positions corresponding to positions 118-120        of SEQ ID NO: 1;    -   ii) a GXSQG motif at positions corresponding to positions        186-190 of SEQ ID NO:1; and    -   iii) an HE motif at positions corresponding to positions 303-304        of SEQ ID NO:1; and

(b) at least one acyl donor substrate, said substrate selected from thegroup consisting of:

-   -   i) esters having the structure

[X]_(m)R₅

-   -   wherein X=an ester group of the formula R₆C(O)O    -   R₆=C1 to C7 linear, branched or cyclic hydrocarbyl moiety,        optionally substituted with hydroxyl groups or C1 to C4 alkoxy        groups, wherein R₆ optionally comprises one or more ether        linkages for R₆=C2 to C7;    -   R₅=a C1 to C6 linear, branched, or cyclic hydrocarbyl moiety or        a five-membered cyclic heteroaromatic moiety or six-membered        cyclic aromatic or heteroaromatic moiety optionally substituted        with hydroxyl groups; wherein each carbon atom in R₅        individually comprises no more than one hydroxyl group or no        more than one ester group or carboxylic acid group; wherein R₅        optionally comprises one or more ether linkages;    -   M is an integer ranging from 1 to the number of carbon atoms in        R₅; and    -   wherein said esters have solubility in water of at least 5 ppm        at 25° C.;    -   ii) glycerides having the structure

-   -   wherein R₁=C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₃ and R₄ are individually H or R₁C(O);    -   iii) one or more esters of the formula

-   -   wherein R₁ is a C1 to C7 straight chain or branched chain alkyl        optionally substituted with an hydroxyl or a C1 to C4 alkoxy        group and R₂ is a C1 to C10 straight chain or branched chain        alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl,        heteroaryl, (CH₂CH₂O)_(n), or (CH₂CH(CH₃)—O)_(n)H and n is 1 to        10; and    -   iv) acetylated saccharides selected from the group consisting of        acetylated monosaccharides, acetylated disaccharides, and        acetylated polysaccharide; wherein upon hydration of the        hydratable adhesive film hydrogen peroxide is released from the        granular bleaching ingredient and said enzyme catalyzes the        formation of an effective amount of a peracid.

Preferred Embodiment 2

The tooth whitening strip according to preferred embodiment 1 whereinthe enzyme having perhydrolytic activity comprises an amino acidsequence selected from:

a) SEQ ID NO: 1; and

b) an amino acid sequence having at least 80% amino acid sequenceidentity to SEQ ID NO: 1.

Preferred Embodiment 3

The tooth whitening strip according to Preferred Embodiment 1 whereinthe enzyme having perhydrolytic activity further comprises a bindingdomain fused to the N- or C-terminus of the enzyme, said binding domainhaving affinity for an oral tissue or for the tooth whitening strip.

Preferred Embodiment 4

The tooth whitening strip according to Preferred Embodiment 3 whereinthe binding domain having affinity for an oral tissue comprises an aminoacid sequence selected from the group consisting of SEQ D NOs: 178-197.

Preferred Embodiment 5

The tooth whitening strip according to any of the above preferredembodiments wherein the enzyme having perhydrolytic activity hasaffinity for an oral tissue and comprises an amino acid sequenceselected from

-   -   a) SEQ ID NO: 2, and    -   b) an amino acid sequence having at least 80% amino acid        sequence identity to SEQ ID NO: 2.

Preferred Embodiment 6

The tooth whitening strip according to any of the above preferredembodiments further comprising a backing layer attached to said secondside of the hydratable adhesive film, said backing layer capable ofinhibiting dissolution of the hydratable adhesive film.

Preferred Embodiment 7

The tooth whitening strip according to any of the above preferredembodiments wherein the granular bleaching ingredient is coated with awater soluble coating capable of dissolving upon hydration.

Preferred Embodiment 8

The tooth whitening strip according to any of the above preferredembodiments wherein the granular bleaching ingredient is selected fromsolid peroxides and solid peroxide donors.

Preferred Embodiment 9

The tooth whitening strip according to any of the above preferredembodiments wherein the granular bleaching ingredient is selected fromperoxide salts, peroxide complexes, peroxyphosphate, peroxycarbonate,perborate, peroxysilicate, persulphate salts, calcium peroxyphosphate,sodium perborate, sodium carbonate peroxide, sodium peroxyphosphate,potassium persulfate, hypochlorites, urea peroxide, hydrogen peroxidepolymer complexes, hydrogen peroxide-polyvinyl pyrrolidone polymercomplexes, metal peroxides, zinc peroxide, calcium peroxide, andcombinations thereof

Preferred Embodiment 10

The tooth whitening strip according to any of the above preferredembodiments wherein the granular bleaching ingredient comprises ureaperoxide. In some embodiments, the granular bleaching ingredientcomprises a hydrogen peroxide-polyvinyl pyrrolidone polymer complex. Insome embodiments, the hydrogen peroxide-polyvinyl pyrrolidone polymercomplex is a hydrogen peroxide-crosslinked polyvinyl pyrrolidone polymercomplex.

Preferred Embodiment 11

The tooth whitening strip according to any of the above preferredembodiments wherein the particle size median diameter (D50) of thegranular bleaching ingredient ranged from 10 microns to 300 microns,e.g., 10 microns to 200 microns.

Preferred Embodiment 12

The tooth whitening strip according to any of the above preferredembodiments wherein the tooth whitening strip comprises from about 0.01wt % to about 0.1 wt % of a peracid. In some embodiments, the granularbleaching ingredient comprises from about 0.1 wt % to about 30 wt % of aperoxygen source.

Preferred Embodiment 13

The tooth whitening strip according to any of the above preferredembodiments wherein the amount of granular bleaching agent on the firstside of the hydratable adhesive film ranges from 0.001 mg/cm² to 10mg/cm², e.g., 0.001 mg/cm² to 1 mg/cm².

Preferred Embodiment 14

The tooth whitening strip according to any of the above preferredembodiments wherein the acyl donor substrate is 1,2,3-triacetoxypropane.

Preferred Embodiment 15

The tooth whitening strip according to any of the above preferredembodiments wherein the hydratable adhesive film comprises one or morewater-soluble polymers selected from a hydrophilic cellulose ether,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, a polyvinyl acetate, a carbomer, a polysaccharide gum,xanthan gum, a modified food starch, gelatin, animal or fish-basedgelatin, a cross-linked carboxyvinyl copolymer, a cross-linkedpolyvinylpyrrolidone, polyethylene oxide, a polyacrylic acid, apolyacrylate, a polyvinyl alcohol, alginate, casein, pullulan, and acombination of two or more thereof.

Preferred Embodiment 16

The tooth whitening strip according to any of the above preferredembodiments wherein the hydratable adhesive film comprises one or morewater-soluble polymers selected from a hydrophilic cellulose ether, apolyvinyl acetate, a carbomer, and a combination of two or more thereof

Preferred Embodiment 17

The tooth whitening strip according to any of the above preferredembodiments wherein the hydratable adhesive film compriseshydroxypropylmethyl cellulose (HPMC), polyvinyl acetate (PVAc), and acarbomer in a dry weight ratio for HMPC:PVAc:carbomer of 10-20:2-10:1.

Preferred Embodiment 18

The tooth whitening strip according to any of the above preferredembodiments wherein the hydratable adhesive film further comprises aplasticizer.

Preferred Embodiment 19

The tooth whitening strip according to any of the above preferredembodiments further comprising propylene glycol.

Preferred Embodiment 20

A method of whitening teeth comprising

a) providing a packaging system comprising the tooth whitening stripaccording to any of the above preferred embodiments;

b) removing the tooth whitening strip form the packaging system; and

c) contacting the tooth whitening strip directly to the teeth for aperiod of time sufficient time whiten the teeth; wherein the toothwhitening strip is hydrated by moisture present in the oral cavity or onthe tooth surface or is hydrated after step (b) but prior to step (c).

Preferred Embodiment 21

The method of Preferred Embodiment 20 wherein the whitening stripfurther comprises a backing layer attached to said second side of thehydratable adhesive film, said backing layer capable of inhibitingdissolution of the hydratable adhesive film.

Preferred Embodiment 22

The method according to any of the above preferred embodiments whereinthe particle size median diameter (D50) of the granular bleachingingredient ranged from 10 microns to 200 microns.

Preferred Embodiment 23

The method according to any of the above preferred embodiments whereinthe granular bleaching ingredient comprises greater than 0.01 wt % ofthe total weight of the hydratable adhesive film and a granularbleaching ingredient attached thereto. In some embodiments, the granularbleaching ingredient comprises greater than 0.05 wt % of the totalweight of the hydratable adhesive film and a granular bleachingingredient attached thereto. In some embodiments, the granular bleachingingredient comprises from about 0.01 wt % to about 0.1 wt % of the totalweight of the hydratable adhesive film and a granular bleachingingredient attached thereto.

Preferred Embodiment 24

The method according to any of the above preferred embodiments whereinthe amount of granular bleaching agent on the first side of thehydratable adhesive film ranges from 0.001 mg/cm² to 1 mg/cm².

Preferred Embodiment 25

The method according to any of the above preferred embodiments whereinthe acyl donor substrate is 1,2,3-triacetoxypropane.

Preferred Embodiment 26

The method according to any of the above preferred embodiments whereinthe hydratable adhesive film comprises one or more water-solublepolymers selected from a hydrophilic cellulose ether, carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, apolyvinyl acetate, a carbomer, a polysaccharide gum, xanthan gum, amodified food starch, gelatin, animal or fish-based gelatin, across-linked carboxyvinyl copolymer, a cross-linkedpolyvinylpyrrolidone, polyethylene oxide, a polyacrylic acid, apolyacrylate, a polyvinyl alcohol, alginate, casein, pullulan, and acombination of two or more thereof

Preferred Embodiment 27

The method according to any of the above preferred embodiments whereinthe hydratable adhesive film comprises one or more water-solublepolymers selected from a hydrophilic cellulose ether, a polyvinylacetate, a carbomer, and a combination of two or more thereof.

Preferred Embodiment 28

The method according to any of the above preferred embodiments whereinthe hydratable adhesive film comprises hydroxypropylmethyl cellulose(HPMC), polyvinyl acetate (PVAc), and a carbomer in a dry weight ratiofor HMPC:PVAc:carbomer of 10-20:2-10:1.

Preferred Embodiment 29

The method according to any of the above preferred embodiments whereinthe hydratable adhesive film further comprises a plasticizer.

Preferred Embodiment 30

The method according to any of the above preferred embodiments whereinthe hydratable adhesive film further comprises propylene glycol.

Preferred Embodiment 31

The method according to any of the above preferred embodiments whereinthe granular bleaching ingredient is coated with a water soluble coatingcapable of dissolving upon hydration.

Preferred Embodiment 32

A method of making a tooth whitening strip comprising:

a) providing a semi-dry hydratable adhesive film,

b) applying to one surface of the film granules of a granular bleachingingredient whereby the granules adhere to the surface, and

c) drying the film.

In some embodiments, the particle size median diameter (D50) of thegranular bleaching ingredient is from 10 microns to 300 microns. In someembodiments, the particle size median diameter (D50) of the granularbleaching ingredient is from 25 microns to 200 microns. In someembodiments, the particle size median diameter (D50) of the granularbleaching ingredient is from 35 microns to 150 microns. In someembodiments, the particle size median diameter (D50) of the granularbleaching ingredient is from 50 microns to 125 microns. In someembodiments, the particle size median diameter (D50) of the granularbleaching ingredient is from 60 microns to 100 microns. In someembodiments, the particle size median diameter (D50) of the granularbleaching ingredient is about 64 microns. In some embodiments, theparticle size median diameter (D50) of the granular bleaching ingredientis about 94 microns.

In some embodiments, the particle size median diameter (D50) of theenzyme having perhydrolytic activity is from 100 microns to 300 microns.In some embodiments, the particle size median diameter (D50) of theenzyme having perhydrolytic activity is from 150 microns to 275 microns.In some embodiments, the particle size median diameter (D50) of theenzyme having perhydrolytic activity is from 175 microns to 250 microns.

All ingredients for use in the strips described herein should be orallyacceptable. By “orally acceptable” as the term is used herein is meantan ingredient which is present in a strip as described in an amount andform which does not render the strip unsafe for use in the oral cavity.

As used throughout, ranges are used as shorthand for describing each andevery value that is within the range. Any value within the range can beselected as the terminus of the range. In addition, all references citedherein are hereby incorporated by referenced in their entireties. In theevent of a conflict in a definition in the present disclosure and thatof a cited reference, the present disclosure controls.

Unless otherwise specified, all percentages and amounts expressed hereinand elsewhere in the specification should be understood to refer topercentages by weight. The amounts given are based on the active weightof the material.

EXAMPLES

The following examples are provided to demonstrate preferred aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples follow techniques tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the presently disclosed methodsand examples.

All reagents and materials were obtained from DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), TCI America (Portland,Oreg.), Roche Diagnostics Corporation (Indianapolis, Ind.), ThermoScientific (Pierce Protein Research Products) (Rockford, Ill.) orSigma/Aldrich Chemical Company (St. Louis, Mo.), unless otherwisespecified.

The following abbreviations in the specification correspond to units ofmeasure, techniques, properties, or compounds as follows: “sec” or “s”means second(s), “min” means minute(s), “h” or “hr” means hour(s), “μL”means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM”means millimolar, “M” means molar, “mmol” means millimole(s), “ppm”means part(s) per million, “wt” means weight, “wt %” means weightpercent, “g” means gram(s), “mg” means milligram(s), “μg” meansmicrogram(s), and “ng” means nanogram(s).

Example 1

A strip is prepared as described above, forming the hydratable adhesivefilm and then while the film is still tacky, adding the granulatedwhitening agent and granulated enzyme to the surface of one side, usingthe ingredients in Table 1. The strip will erode slowly in the mouthupon application, and so does not need to be removed.

TABLE 1 Dry Strip Concentration Ingredients in Strip (wt %)Hydroxypropylmethylcellulose (HPMC) 59 Polyvinylacetate (PVAc) 30Carbopol, 971 5 Triacetin 5 Titanium dioxide 1 Total 100 ConcentrationIngredients in Bleach Granule (wt %) Urea peroxide 100 ConcentrationIngredients in Enzyme Granule (wt %) Cornstarch 99 Perhydrolase enzyme 1

The strip in example 1 is cast from water by extrusion, or by castingfrom a water suspension (for example at a solids level of 10-30%) onto aheated belt, from which the water is evaporated. The granules are addedto the surface of this film when the film is semi-dry or dry, but stilltacky. Once cooled to room temperature, the granules adhere to thesurface of the film. For a strip with an area of 10 cm² and a weight of10 mg/cm², this formula delivers 5 mg triacetin. Assuming that 1.4 mg ofbleach granules are distributed on the strip, along with 0.3 mg ofenzyme granules, this dose is sufficient to produce enough peraceticacid directly at the tooth surface to significantly outperform aperoxide-only whitening strip. (Peroxide only strips typically have atotal dose of approximately 3-10 mg peroxide.)

When exposed to saliva or other sources of water (such as tap water),the granules immediate dissolve and become active. The adhesive layeralso is activated and sticks to the teeth effectively. Example 1 isdesigned to slowly erode in the mouth over time, so the user does notneed to remove it.

Example 2

A strip is prepared as described above, forming the hydratable adhesivefilm and then while the strip is still tacky, adding the granulatedagents to one side and the protective backing layer to the other side,using the ingredients in Table 2. Because the backing layer will notdissolve, the user should remove it after a sufficient period has passedto permit whitening to take place, typically about 10-30 minutes. Thetwo layers can also be produced simultaneously by extrusion orsolvent-based casting, then the granulated whitening agent can be addedto the surface of the hydratable adhesive film.

TABLE 2 Dry Strip Concentration Ingredients in Layer #1 (wt %) Ethylcellulose 94 Propylene glycol 5 Titanium dioxide 1 Total 100 Dry StripConcentration Ingredients in Layer #2 (wt %)Hydroxypropylmethylcellulose 69 (HPMC) Polyvinylacetate (PVAc) 15Carbopol, 971 5 Triacetin 5 Titanium dioxide 1 Flavor 5 Total 100Concentration Ingredients in Bleach Granule (wt %) Gum arabic 10 Ureaperoxide 90 Concentration Ingredients in Enzyme Granule (wt %)Cornstarch 99 Perhydrolase enzyme 1

Example 3

Various particle sizes of hydrogen peroxide-polyvinylpyrrolidone complexand an enzyme having perhydrolytic activity (“enzyme”) were evaluatedfor their ability to generate peracetic acid uniformly across thesurface of a hydratable adhesive strip containing triacetin.

To evaluate the generation of peracetic acid from this product, ⅜″ discswere cut from the film. Each disc was hydrated with 20 μL of 50 mMsodium phosphate buffer, pH 7.2 and incubated at 37° C. for 15 min. 380μL of 0.1 M phosphoric acid was added to the film to quench the enzymereaction and dilute the sample for detection. The solution was analyzedfor peracetic acid with HPLC analyses for peracetic acid using themethod described previously in U.S. Pat. No. 7,829,315 to DiCosimo etal. For each evaluation, a minimum of three samples were cut from thestrip product across a 24 inch length of film.

Strip samples with the enzyme and hydrogen peroxide-polyvinylpyrrolidonecoating were made by first incorporating the enzyme in a pullulanpolymer film. The dried film containing the enzyme was milled and sievedto a particle size of 60 to 80 mesh (177 μm to 250 μm). The particulateform of enzyme was then blended with hydrogenperoxide-polyvinylpyrrolidone (PEROXYDONE™ XL-10, Ashland Inc.,Wilmington, Del.). This blend was deposited onto a two-layer filmstructure with a hydratable adhesive layer containing primarilypolyethylene oxide and triacetin and a backing layer of polyvinylalcohol to provide a non-dissolvable support layer. The results ofevaluation of peracetic acid generation for two consecutive runs arelisted in Table 3. Samples were evaluated at the beginning and end ofthe production run and demonstrate poor consistency for peracetic acid.

TABLE 3 Peracetic acid production from hydratable adhesive stripsproduced with a coating of perhydrolase enzyme in pullulan andPEROXYDONE ™ XL-10. Start of Run End of Run Std Dev Avg PAA Std DevSample Avg PAA (ppm) (3 replicates) (ppm) (3 replicates) 1 304 27 110 52 132 16 55 3

The PEROXYDONE™ XL-10 was further processed to form larger particleswith ethanol high shear granulation. After granulation the sample wassieved to a particle size of 60 to 200 mesh (75 μm to 250 μm). Theparticle size distribution of PEROXYDONE™ XL-10, as used to producesamples described in Table 3, and the PEROXYDONE™ XL-10 following highshear granulation and sieving are described in Table 4. The samples weremeasured on a Beckman Coulter LS13320 equipped with a Tornado dryfeeder. The powders were analyzed directly without being dispersed in aliquid.

TABLE 4 Particle size distribution for PEROXYDONE ™ XL-10 used indeposition before and after granulation. Particle Size DistributionPEROXYDONE ™ Statistics (μm) XL-10 Sample Mean Median D10 D90 Asreceived 75.9 64.0 28.5 143.5 After granulation 100.5 93.8 49.9 165.1

Another strip production run was completed by first preparing an enzymesample loaded into the pullulan matrix at twice the concentration ofsamples from Table 3. The pullulan film was milled and sieved to 60 to80 mesh (177 μm to 250 μm) and then combined with the granulatedPEROXYDONE™ XL-10. The blend was then coated onto a similar two-layerfilm structure as described above. The evaluation of this sample forperacetic acid generation is provided in Table 4, and demonstratedhigher and more consistent production of peracetic acid throughout theproduction run.

TABLE 5 Peracetic acid production from hydratable adhesive stripsproduced with a coating of perhydrolase enzyme in pullulan and highshear granulated PEROXYDONE ™ XL-10. Start of Run End of Run Std Dev AvgPAA Std Dev Sample Avg PAA (ppm) (3 replicates) (ppm) (3 replicates) 3828 51 1039 162

1. A tooth whitening strip comprising a hydratable adhesive film with afirst side and a second side, the first side having a granular bleachingingredient attached thereto, wherein the strip comprises, in or on thefilm or in the form of granules on the first side of the film, (i) aprotein having perhydrolase activity which contains the catalytic domainof a member of the carbohydrate esterase family 7; and (ii) a carboxydonor, wherein upon use, the peroxide released by the granular bleachingingredient reacts with the carboxy donor in the presence of theperhydrolase to form a peracid.
 2. The tooth whitening strip accordingto claim 1 wherein the protein having perhydrolase activity comprises anamino acid sequence selected from: a) SEQ ID NO: 1MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNISPPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKG,

b) an amino acid sequence having i) an RGQ motif at positionscorresponding to positions 118-120 of SEQ ID NO: 1; ii) a GXSQG motif atpositions corresponding to positions 179-183 of SEQ ID NO: 1; and iii)an HE motif at positions corresponding to positions 298-299 of SEQ IDNO: 1; and c) an amino acid sequence having at least 80%, e.g., at least90%, e.g., at least 95% sequence identity to SEQ ID NO:
 1. 3. The toothwhitening strip according to claim 1 wherein the protein havingperhydrolase activity comprises an amino acid sequence having affinityfor oral tissue or for the whitening strip.
 4. The tooth whitening stripaccording to claim 3 wherein the protein having perhydrolase activityhas affinity for oral tissue and comprises an amino acid sequenceselected from a) SEQ ID NO: 2MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNISPPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKGGPGSGGAGSPGSAGGPGSTKPPRTPTANTSRPHHNFGSGGGGSPHHHHHH, and

b) an amino acid sequence having at least 80%, e.g., at least 90%, e.g.,at least 95% sequence identity to SEQ ID NO:
 2. 5. The tooth whiteningstrip according to claim 1, further comprising a backing layer.
 6. Thetooth whitening strip according to claim 1 wherein the granularbleaching ingredient is coated with a quickly dissolving material. 7.The tooth whitening strip according to claim 1 wherein the granularbleaching ingredient is selected from solid peroxides and solid peroxidedonors.
 8. The tooth whitening strip according to claim 1 wherein thegranular bleaching ingredient is selected from peroxide salts orcomplexes, peroxyphosphate, peroxycarbonate, perborate, peroxysilicate,persulphate salts, calcium peroxyphosphate, sodium perborate, sodiumcarbonate peroxide, sodium peroxyphosphate, potassium persulfate,hypochlorites, urea peroxide, hydrogen peroxide polymer complexes,hydrogen peroxide-polyvinyl pyrrolidone polymer complexes, metalperoxides, zinc peroxide, calcium peroxide, peracids, and combinationsthereof.
 9. The tooth whitening strip according to claim 1 wherein thegranular bleaching ingredient comprises urea peroxide.
 10. The toothwhitening strip according to claim 3 foregoing claims the particle size(D50) of the granular bleaching ingredient is 10-200 microns.
 11. Thetooth whitening strip according to claim 10 wherein the granularbleaching ingredient comprises 0.1% or less of the total weight of thehydratable adhesive film and a granular bleaching ingredient attachedthereto.
 12. The tooth whitening strip according to claim 11 wherein theamount of granular bleaching agent on the first side of the hydratableadhesive film is 0.001-1 mg/cm².
 13. The tooth whitening strip accordingto claim 12 wherein the carboxy donor is selected from (i) one or moreC2-18 carboxylic acids, optionally substituted with hydroxy and/or C1-4alkoxy; (ii) one or more hydrolysable and acceptable esters thereof, and(iii) mixtures thereof.
 14. The tooth whitening strip according to claim13 wherein the carboxy donor is 1,2,3-triacetoxypropane.
 15. The toothwhitening strip according to claim 1 which comprises a peracid or whichgenerates a peracid upon use.
 16. The tooth whitening strip according toclaim 1 wherein the ingredients are present in amounts sufficient toprovide, upon mixing, a bleaching agent in an amount and concentrationeffective to whiten teeth.
 17. The tooth whitening strip according toclaim 1 wherein the hydratable adhesive film comprises one or morewater-soluble polymers selected from a hydrophilic cellulose ether,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, a polyvinyl acetate, a carbomer, a polysaccharide gum,xanthan gum, a modified food starch, gelatin, animal or fish-basedgelatin, a cross-linked carboxyvinyl copolymer, a cross-linkedpolyvinylpyrrolidone, polyethylene oxide, a polyacrylic acid, apolyacrylate, a polyvinyl alcohol, alginate, casein, pullulan, and acombination of two or more thereof.
 18. The tooth whitening stripaccording to claim 1, wherein the hydratable adhesive film comprises oneor more water-soluble polymers selected from a hydrophilic celluloseether, a polyvinyl alcohol, a carbomer, and a combination of two or morethereof.
 19. The tooth whitening strip according to claim 1, wherein thehydratable adhesive film further comprises a plasticizer.
 20. The toothwhitening strip according to claim 1, wherein the hydratable adhesivefilm further comprises propylene glycol.
 21. The tooth whitening stripaccording to claim 1, wherein the granular bleaching ingredient iscoated with a water soluble coating capable of dissolving uponhydration.
 22. The tooth whitening strip according to claim 1, whereinan effective amount of a peracid is generated at a substantially uniformconcentration across the surface of the tooth whitening strip.
 23. Amethod of whitening teeth comprising a) providing a packaging systemcomprising the tooth whitening strip according to any foregoing claim;b) removing the tooth whitening strip form the packaging system; and c)contacting the tooth whitening strip directly to the teeth for a periodof time sufficient time whiten the teeth; wherein the tooth whiteningstrip is hydrated by moisture present in the oral cavity or on the toothsurface or is hydrated after step (b) but prior to step (c).